Delivery system to modulate immune response

ABSTRACT

A microsphere containing an immunogen bound to an inert particle having a mesh size of greater than about 35 mesh for site-specific release and induction of an immune response. The immune response may be an overall enhanced T lymphocyte immune response or a selective response. The physical and chemical characteristics and/or modes of administration of the microsphere may be engineered to increase T H 1 lymphocytes for treatment of cancer or infectious disease. The microencapsulated immunogen has an enteric coating for oral administration.

FIELD OF THE INVENTION

[0001] This invention is directed generally to a method of selectingand/or selectively modulating an immune response by administering amicroencapsulated immunogen.

BACKGROUND OF THE INVENTION

[0002] The immune system recognizes and distinguishes substances as selfversus nonself, and defends the body against nonself substances. Theimportance of this distinction is evident in a variety of conditionssuch as autoimmune diseases, rejection of transplanted tissues ororgans, allergic reactions, cancer and infectious diseases, and modes oftreatments such as immunotherapy and gene therapy. For example, inautoimmune diseases such as rheumatoid arthritis, systemic lupuserythematosus and myasthenia gravis, the body mistakenly treats self asnonself and thus destroys its own components. In transplant rejection,immunosuppressive drugs are administered to a recipient to prevent therecipient's immune system from rejecting a true nonself substance sothat the recipient can accept the transplanted tissue or organ as itsown. In allergic reactions such as asthma, eczema and hay fever, thereis an immune hypersensitivity in some individuals that occursimmediately following contact with an antigen. In infectious diseases amicrobe such as a bacterium, parasite or virus stimulates an immuneresponse. The microbe or a microbe subunit may be formulated as avaccine to provide prophylactic protection against subsequent infection.In cancer, unlike the other conditions, an immune response is notmounted and the lack of an immune response plays a role in theuncontrolled growth of malignant cells. A wide variety of foreignsubstances, termed antigens or immunogens, elicit an immune response andthus are targeted by the immune system. Examples of antigens include,but are not limited to, infectious disease agents such as bacteria,viruses, parasites and fungi as well as mites, pollen, animal dander,drugs, toxins and chemicals.

[0003] The immune system is a complex network of cells, tissues andorgans that directly and indirectly target and ultimately destroyforeign substances. Of the various cells involved in mounting an immuneresponse, lymphocytes are one type of white blood cells that have acrucial role. One type of lymphocyte is the B lymphocyte (B cell) thattargets and indirectly destroys foreign substances by mounting a humoralimmune response to produce antibodies against specific antigens. Theother type of lymphocyte is the T lymphocyte (T cell) that targets anddirectly kills foreign substances by mounting a cell-mediated immuneresponse. There are three major subtypes of T cells designated as Thelper cells, T suppressor cells, and T cytotoxic cells. T helper cellsare of two types: T_(H)1 and T_(H)2 cells. T_(H)2 cells help B cellsmount a humoral immune response and help T cytotoxic cells maintainthemselves by producing growth factors needed by the T cytotoxic cells.T_(H)2 cells express the CD4 glycoprotein antigen. T suppressor cellsinhibit or suppress T helper cells; they express the CD8 glycoproteinantigen. T cytotoxic cells, also called cytotoxic T lymphocytes (CTL),express the CD8 glycoprotein antigen and are a subset of T cells thatkill cells expressing a specific antigen upon direct contact with thesetarget cells. Pre-CTL are T cells that are committed to the CTL lineage,have undergone thymic maturation and are already specific for aparticular antigen, but lack cytolytic function. CTL are importanteffector cells in three settings: (1) intracellular infections ofnon-phagocytic cells or infections that are not completely contained byphagocytosis such as viral infections, (2) infections by bacteria suchas Listeria monocytogenes, and (3) acute allograft rejection andrejection of tumors.

[0004] An immunogenic response is most predictably induced by using aprotein as the immunogen. In immunotherapy, the protein is frequentlyadministered parenterally, for example by injection. While injectionsare inconvenient and uncomfortable to many patients, they haveheretofore been a common route of administration because orallyadministered protein is degraded by protease enzymes and acid in thestomach and enzymes in the small intestines. It has been demonstratedthat oral administration of a soluble protein such as the model antigenovalbumin (OVA) results in the induction of immune tolerance,characterized by the loss of either antibody or T cell response to theprotein antigen. However, U.S. Pat. No. 5,591,433 discloses thatimmunologically active biomolecules and other therapeutic proteins canbe orally administered by microencapsulating the protein and coating themicrosphere to form a pH-sensitive enterocoated microsphere particlethat is resistant to the action of digestive proteolytic enzymes andacids. The microspheres disclosed in the '433 patent consist of proteinbound to an inert particle having a mesh size of about 30-35 mesh (about600 μm to about 500 μm) diameter and coated with an acid stable polymer.What is needed, however, is a method of better selecting and selectivelymodulating a particular immune response from the complex immunerepertoire to better respond to different antigenic stimuli in differentconditions requiring treatment.

[0005] For example, current cancer treatments include combinations ofchemotherapy, radiation therapy, and surgical excision of some or all ofa solid tumor. Each of these treatment mechanisms is targeted toeliminating malignant cells but is performed at the expense ofdestroying nonmalignant cells. Thus, none of these treatments utilizethe body's own capacity for cell destruction, namely, the immune systemand particularly the cytotoxic T cells, to kill malignant cells. Amethod of increasing an immune response and/or selectively stimulatingthe cytotoxic T cell population would therefore be a valuable supplementto traditional treatment methods. In addition, such a method wouldoperate without the adverse effects of chemotherapeutic drugs,radiation, or surgical insult. Cancer cells, however, are not recognizedas foreign by the immune system and thus are not targeted fordestruction. One goal in developing cancer treatments is to stimulatethe immune system to mount an immune response against cancer cells. Ofthe three major T cell types, the T cytotoxic cells frequently directlytarget and destroy cancer cells. Thus, selectively increasing the Tcytotoxic cell subtype may be an advantageous way to check theunregulated cell division that is a hallmark of cancer cells.

[0006] As another example, the T cytotoxic cells also directly targetand destroy extracellular infectious disease agents and infectiousdisease agents in infected cells. Cell mediated immunity consists of twotypes of reactions.

[0007] The first type is macrophage activation resulting in the killingof phagocytized microbes. The second type is lysis of infected cells byCD8+ cytotoxic T lymphocytes (CTL). Differences among individuals in thepatterns of immune responses to intracellular microbes, for example inHIV infection, are important determinants of disease progression andclinical outcome. The selective increase in the T cytotoxic cell subtypemay be used to combat infectious diseases.

[0008] There is thus a need for a method and composition to bettermodulate and/or selectively stimulate an immune response. Such a methodand composition would find wide use in immunotherapy or gene therapy forconditions such as allergies, infectious diseases, cancer, transplantrejection, and autoimmune diseases. Such a method and composition wouldalso be a valuable prophylactic and/or therapeutic supplement to currentmethods of treating these conditions.

SUMMARY OF THE INVENTION

[0009] This invention provides methods and compositions to induce anenhanced general or selective immune response. A drug delivery systemcomprises a microsphere of an immunogen bound to an inert particlehaving a mesh size greater than about 35 mesh. The microsphere isadministered to the small intestine of a mammal. The microsphere ispreferably administered orally and contains one or more enteric coatingsand may be administered in a gel capsule. In one embodiment the inertparticle has a mesh size greater than about 40 mesh and may be anonpareil, a silica powder, a salt crystal or a sugar crystal.

[0010] The response may encompass a general enhanced production ofT_(H)1 cells, T_(H)2 cells and cytotoxic T lymphocyte (CTL) subsets, oran enhanced shift from a T_(H)2 type response to a T_(H)1 type response,or an enhanced shift from a T_(H)1 type response to a T_(H)2 typeresponse, or an enhanced differentiation of pre-CTL to CTL. Theimmunogen may be a peptide, a protein fragment, a protein, a DNA, and/oran RNA, and may be a gene, a gene fragment or a vaccine.

[0011] The immunogen may be administered in a dosing regimen and/or adosing composition containing a number of microspheres to selectivelyinduce a particular immune response. The microspheres of the dose maycontain the same enteric coatings or different enteric coatings, thesame formulation or different formulations, and/or the same inertparticle core composition and size or different core compositions andsizes. The immunogen may also be administered with a potentiating agent,either in a single inert particle or in separate inert particles. Ifformulated with the immunogen and potentiating agent in a single inertparticle, the various single inert particles of the administered dosemay have the same enteric coating or a different enteric coating, thesame formulations or different formulations, and/or the same inertparticle core composition and size or different core compositions andsizes. Likewise, if formulated with the immunogen and potentiating agentin separate inert particles, the separate microspheres of theadministered dose may have the same enteric coatings or differententeric coatings, the same formulations or different formulations,and/or the same inert core compositions and sizes or different corecompositions and sizes.

[0012] As will be appreciated, the disclosed delivery system and methodsof using the system have a wide array of applications. These and otheradvantages of the invention will be further understood with reference tothe following drawings, detailed description and examples.

BRIEF DESCRIPTION OF THE DRAWINGS

[0013]FIG. 1 is a graph of the results of primary lymphocyteproliferation with different modes of ovalbumin (OVA) administration.

[0014]FIG. 2A is a graph of the results of a lymphoproliferativeanalysis using either microspheres containing OVA, OVA in adjuvant, orplacebo microspheres, and

[0015]FIG. 2B is a graph of the results using concanavalin A (Con A)nonspecific mitogen stimulation.

[0016]FIG. 3A is a graph of the results from in vitro stimulation withmicrospheres containing OVA, and

[0017]FIG. 3B is a graph of the results using Con A nonspecific mitogenstimulation.

[0018]FIG. 4 is a graph of cytotoxic T lymphocyte responses at differenteffector:target ratios.

[0019]FIG. 5A is a graph of the results of antibody blocking experimentsfor microspheres containing OVA, and

[0020]FIG. 5B is a graph of the results using Con A nonspecific mitogenstimulation.

DETAILED DESCRIPTION OF THE INVENTION

[0021] Definition of Terms

[0022] The terms immunogen or antigen are broadly used herein toencompass any chemical or biological substance that elicits an immuneresponse when administered to a mammal. While an immunogen is frequentlya protein, it may also be a nucleic acid. For the purpose of the presentinvention, immunogens include but are not limited to the following:allergenic proteins and digested fragments thereof such as pollenallergens from ragweed, rye, June grass, orchard grass, sweet vernalgrass, red top grass, 15 timothy grass, yellow dock, wheat, corn,sagebrush, blue grass, California annual grass, pigweed, Bermuda grass,Russian thistle, mountain cedar, oak, box elder, sycamore, maple, elmand so on, dust, mites, bee and other insect venoms, food allergens,animal dander, microbial vaccines which in turn include viral,bacterial, protozoal, nematode and helminthic vaccines and their variouscomponents such as surface antigens, including vaccines which containglycoproteins or proteins, protein fragments, genes or gene fragmentsprepared from, for example, Staphylococcus aureus, Streptococcuspyogenes, Streptococcus pneumoniae, Neisseria meningitidis, Neisseriagonorrhoeae, Salmonellae species, Shigellae species, Escherichia coil,Klebsiellae species, Proteus species, Vibrio cholerae, Helicobacterpylori, Pseudomonas aeruginosa, Haemophilus influenzae, Bordetellapertussis, Mycobacterium tuberculosis, Legionella pneumophila, Treponemapallidum, and Chlamydiae species, tetanus toxoid, diphtheria toxoid,influenza viruses, adenoviruses, paramyxoviruses, rubella viruses,polioviruses, hepatitis viruses, herpesviruses, rabies viruses, humanimmunodeficiency viruses, and papilloma viruses, in addition toprotozoal parasites such as Toxoplasma gondii, Pneumocystis carinii,Giardia lamblia, Trichomonas vaginalis, Isospora beeli, Balantidiumcoli, Blastocystis hominis, and the various species of Entamoeba,Amebae, Plasmodium, Leishmania, Trypanosoma, Babesia, Cryptosporidium,Sarcocystis, and Cyclospora, as well as nematodes and helminths of thevarious species of trematodes, flukes, cestodes and visceral larvae.

[0023] Immunogens may be administered as therapeutic or prophylacticagents, either with or without a potentiating agent. A therapeuticimmunogen is defined herein as one that alleviates a pathologicalcondition or disease. Therapeutic agents that may be used in the presentinvention include, but are not limited to, immunogenic agents and genetherapy agents. A prophylactic agent is defined herein as one thateither prevents or decreases the severity of a subsequently acquireddisease or pathological process. An example of a prophylactic agent is avaccine against a microbe causing an infectious disease. A potentiatingagent is defined herein as one that enhances the antigenicity of otherimmunogens. A potentiating agent thus indirectly stimulates an immuneresponse. An example of a potentiating agent is an adjuvant, definedherein as any biological or chemical substance which, when administeredwith an immunogen, enhances the immune response against the immunogen.Examples of adjuvants are inorganic salts such as aluminum hydroxide(alum), cytokines, and bacterial endotoxins such as cholera toxin B(CTB). Another example of a potentiating agent is a hapten, definedherein as a low molecular weight substance that itself isnon-immunogenic but becomes immunogenic when conjugated to a highmolecular weight carrier. Other potentiating agents includebioadhesives, mucoadhesives and promoting agents.

[0024] Microsphere Formulations

[0025] As used herein and unless specifically indicated otherwise, allpercentages are given in terms of the weight of the ingredient relativeto the total weight of the microsphere. In one embodiment of theinvention, an aqueous solution of the immunogen with an optionalstabilizing agent to provide physical protection for the immunogen isformed. The aqueous immunogen solution will generally be from about 0.5%to about 10% by weight of the immunogen in the microsphere, with about1% being preferred.

[0026] Stabilizing agents are generally therapeutically inactive, watersoluble sugars that act to protect the immunogen during a step in theformulation of the immunogen and/or during a subsequent coating step.Examples of stabilizing agents include the sugars lactose, mannitol andtrehalose. The stabilizing agent is added at a concentration of fromabout 0.1% to about 10%, with a concentration of about 5% beingpreferred. If the immunogen solution has a low viscosity, it may bedesirable to add from about 1% to about 10% of polyvinyl pyrrolidone orother binding agents such as hydroxypropylcellulose orhydroxypropylmethylcellulose to bind the immunogen to the inertparticle.

[0027] The solution of one or more immunogens and an optionalstabilizing agent is then applied, for example by spraying, to apharmaceutically inert material substrate, hereinafter termed an inertparticle. The inert particle may encompass a variety of shapes and formssuch as a bead, a sphere, a powder, a crystal, or a granule. In oneembodiment, a nonpareil, defined as a small round particle of apharmaceutically inert material, may be used. One such nonpareil isavailable under the brand name NuPareils® (Crompton & Knowles Corp.,Mahwah, N.J.). In other embodiments, a silica powder, sugar crystal orsalt crystal may be used. The inert particle in whatever shape or formhas a mesh size greater than about 35 mesh, preferably greater thanabout 40 mesh, and most preferably in the range of about 45 to 200 mesh.

[0028] Glatt® brand powder coater granulators such as the GPCG-1 HS,GPCG-5HS, or GPCG-60HS fluid bed coaters are suitable for use to coatthe immunogen onto the inert particle. Various other brands of Wurstertype fluid bed coaters (NIRO, Vector, Fluid Air, etc.) are also suitablefor use. Coating conditions and times vary depending on the apparatusand coating viscosity; however, coating must generally be conducted attemperatures less than about 50° C., and preferably less than about 35°C., to avoid denaturation of a protein immunogen.

[0029] The dry immunogen-coated inert particles are preferably alsocoated with one or more layers of acid stable polymers to form anenteric coating. This coating renders the immunogen resistant todegradation in the acid environment of the stomach. In addition, varyingthe composition and/or amount of the enteric coating may allow theenteric coating to dissolve, and thus release the immunogen, at aparticular pH in the small intestine for an optimally selective T cellresponse. The coating of one or more polymers may be applied in asimilar manner and with similar equipment as the coating stepspreviously described.

[0030] The enteric coating is preferably a water-based emulsion polymersuch as ethylacrylate methacrylic acid copolymer, sold as Eudragit®L-30D (Hüls America Inc., Somerset, N.J.) with a molecular weight ofabout 250,000 and generally applied as a 30%^(wv) aqueous dispersion.Some examples of alternative polymer coatings are the solvent freeEudragit L/S 100 or hydroxypropylmethyl cellulose acetate succinate. Theenteric coating allows the microencapsulated immunogen to be orallyadministered without being released from the microsphere untilencountering a specific region of the gut. The chemical composition ofthe enteric coating may be formulated to dissolve, and thus release theimmunogen, at a particular pH in the small intestine for an optimallyselective T cell response. Alternatively, the enteric coating may beformulated to release the immunogen after encountering sufficientmechanical and/or chemical erosion.

[0031] The coating composition may be combined with a plasticizer toimprove the continuity of the coating. Several well known plasticizersmay be used, with triethylcitrate (Morflex Inc., Greensboro, N.C.)preferred. Although plasticizers can be liquid, they are not consideredto be solvents since they lodge within the coating and alter itsphysical characteristics but do not act to dissolve the proteinimmunogen. A plasticizer which dissolves or denatures the immunogenwould be unacceptable.

[0032] Talc (about 3.0%) may be added to prevent the particles fromsticking to each other. An antifoaming agent (about 0.0025%) such assorbitan sesquioleate (Nikko Chemicals Co. Ltd., Japan) or silicone canalso be added. An antistatic agent (about 0.1%) such as Syloid 74FP(Davison Chemical Division, Cincinnati, Ohio) can be added. The talc,antifoaming agent and antistatic agent are added only if needed.

[0033] The inert particles containing the immunogen, the optionalstabilizing agent or agents and other formulation ingredients are driedand may be coated with the enteric coating as previously described. Thecoating solution is about 30% to about 75% polymer, about 0% to about10% plasticizer, about 0% to about 3% talc, about 0% to about 0.0025%antifoaming agent, about 0% to 3% antistatic agent and water. It isgenerally preferable that there be no organic solvents, includingalcohols and even glycols, present in the coating composition as organicsolvents can denature the immunogen.

[0034] Potentiating Agents

[0035] In an alternative embodiment, a potentiating agent may be addedto increase the immunogenicity of the protein. Examples of potentiatingagents include adjuvants, bioadhesives, mucoadhesives, and promotingagents. Adjuvants work by either concentrating antigen at a site wherelymphocytes are exposed to the antigen or by inducing cytokines whichregulate lymphocyte function. The adjuvant may be either a biologicalcompound, a chemical compound that is therapeutically acceptable, or acombination of a biological and chemical compound. Examples of chemicaladjuvants are water dispersible inorganic salts such as aluminumsulfate, aluminum hydroxide (alum) and aluminum phosphate. Examples ofbiological adjuvants are endogenous cytokines such asgranulocyte-macrophage colony-stimulating factor (GM-CSF), tumornecrosis factor-a (TNF-α), interleukin-2 (IL-2), interleukin-4 (IL-4),interleukin-12 (IL-12) and γ-interferon (IFNγ), microorganisms such asBCG (bacille Calmette-Guerin), Corynebacterium parvum, and Bordetellapertussis, bacterial endotoxins such as cholera toxin B (CTB),lipopolysaccharide (LPS), and muramyldipeptide (N-acetyl-muramyl-L-alanyl-D-isoglutamine [MDP]). Commercially available adjuvants suchas DETOX-PC® are also available. Bioadhesives such as Lycopersiconesculentum lectin (tomato lectin, LT) and mucoadhesives such asChitosans-like N-trimethyl chitosan chloride bind to sugars and formglycoconjugates at site-specific areas of the intestines. Promotingagents are defined herein as formulation ingredient(s) that promoteuptake, transport or presentation of antigen(s), adjuvants, or haptensthereby enhancing the desired immune response. Examples of promotingagents are glycoproteins, lipoproteins, bile salts, fatty acids,phospholipids, glycolipids, triglycerides, and cholesterol,cyclodextrins, glycerol, among others. All of the above potentiatingagents may be incorporated into the microsphere formulation singly, incombination, or as part of covalent or noncovalent complexes.

[0036] The potentiating agent may be added to the aqueous dispersion orsolution of immunogen prior to coating onto the inert particle.Alternatively, the potentiating agent may be added to non-immunogenbound inert particles. Generally, about 1% to about 10% of potentiatingagent is added. The potentiating agent may be bound to the same inertparticle as the immunogen. Alternatively, the potentiating agent may bebound to a first inert particle and the immunogen may be bound to asecond inert particle, such that the potentiating agent may be appliedto non-immunogen bound inert particles.

[0037] Proposed Mechanism of Action

[0038] It has been found that microspheres produced from inert particleshaving a mesh size greater than about 35 mesh enhance and selectivelystimulate T cytotoxic cells over other types of T cells. As shown inFIG. 1, the microspheres of the present invention have a potentiatingagent-like effect and the extent of T cell stimulation increases withdecreasing size of the inert particle of the microsphere. Microspherescontaining OVA with an inert particle mesh size greater than about 35mesh (open bars) stimulated primary lymphocytes more than twice as muchas microspheres containing both OVA and adjuvant with an inert particlemesh size less than about 35 mesh (solid bars). Microspheres containingOVA with a mesh size greater than about 35 mesh stimulated primarylymphocytes more than three times as much as parenterally administeredOVA with adjuvant (hatched bars). This demonstrates that by usingenteric coated immunogens attached to an inert particle having a meshsize greater than about 40 mesh, a potentiating agent-like effect inselecting for a T cytotoxic cell response is produced that is equivalentto the response produced using OVA administered with DETOX-PC® adjuvant.Thus, adding an adjuvant such as aluminum hydroxide (alum) or DETOX-PC®or other potentiating agent(s) to the microsphere formulation in certaincases may provide additional stimulation of a T cytotoxic cellpopulation, and may allow a lower initial dose of immunostimulatory drugto generate an immune response equivalent to that obtained with a higherdose of immunostimulatory drug.

[0039] While the exact mechanism for these selective stimulations isunclear, one explanation may be that smaller enteric antigen coatedparticles provide an increase in contact points between the immunogenencapsulated therein and the appropriate immune cell receptor systemslying along the mammalian intestinal tract, particularly in the diffuselymphatic tissue of Peyer's patches. These smaller particles alsocontain more of certain formulation ingredients on a per weight basis,some of which may enhance antigen presentation and delivery. Otherexplanations, however, may be possible.

[0040] Microsphere Dosing

[0041] In use, the microspheres of the present invention, comprisingimmunogen-bound inert particles having a mesh size greater than about 35mesh and enteric coated with an optional potentiating agent, areadministered in a dosing schedule and composition comprising variouspermutations of the above sizes and compositions to modulate an immuneresponse. The microspheres are preferably administered orally such as bygavage or feeding, or may be administered parenterally such as bysubcutaneous injection. Dosing may be consecutive or intermittent and atvarious times and in various formulations. As used herein, formulationsencompass both the different percentage compositions and differentphysicochemical compositions of the microspheres, such as size,coatings, polymers, plasticizers, anti-stick agents, anti-foam agents,antistatic agents, potentiating agent(s) and excipients.

[0042] For example, an administered dose may contain a number of singleinert particles with each inert particle containing one or moreimmunogens and, if added, the potentiating agent. If formulated as asingle inert particle, the various single microspheres of theadministered dose may have the same enteric coating or different entericcoatings, the same formulation or different formulations of polymers,plasticizers, binding agents, anti-stick agents, anti-foam agents,antistatic agents, potentiating agent(s) and excipients, and/or the sameinert core composition and size or different inert core compositions andsizes. Alternatively, the dose may be formulated to contain acombination of inert particles with one or more immunogens and, ifadded, the potentiating agent(s) in separate inert particles. Ifformulated with the immunogen and potentiating agent(s) in separateinert particles, the separate microspheres of the administered dose mayhave the same enteric coatings or different enteric coatings, the sameformulations or different formulations of polymers, plasticizers,binding agents, anti-stick agents, anti-foam agents, antistatic agents,potentiating agent(s) and excipients, and/or the same inert corecompositions and sizes or different inert core compositions and sizes.These various combinations and permutations of inert particle size,inert particle composition, enteric coating, and formula compositionhelp to achieve selective distribution and presentation of the antigenalong the gut upon administration of the microspheres.

[0043] The microspheres may be placed in gel capsules for oraladministration to humans or other mammals. Dosage will depend on theindividual and the course of the therapy. For example, in treatmentusing the microspheres of the invention containing ragweed as theimmunogen, the dosage would be about 0.03 to about 35 units in terms ofa major allergenic protein, Amb-a-1, administered daily. Dosage forallergens may be different from the dosage used in immunotherapy byinjection.

[0044] Applications

[0045] In use, the microspheres of the present invention containing anenteric coated immunogen and an optional potentiating agent havenumerous applications. For example microspheres containingglycoproteins, proteins, protein fragments, peptides, or gene fragmentsfrom microorganisms, viruses or parasites would be a valuableprophylactic and/or therapeutic supplement to the typical antimicrobial,antiviral and antiparasitic agents administered to treat infectiousdiseases. As another example, a peptide fragment containing nondominantepitope(s) from the HER-2/neu oncogenic “self-protein” can be used asthe immunogen in the microspheres of the invention to increase theefficacy of a cancer vaccine by breaking tolerance against overexpressedtumor proteins. This use would be especially valuable since HER-2/neu isa “self” protein and thus does not generate an immune response. By usinga peptide containing nondominant epitope(s) rather than the wholeprotein as reported by Disis et al. (J. Immunol., 1996:156, 3151-3158)in the microspheres of the invention, a cancer vaccine eliciting a Tcytotoxic cell response targeting “self” tumor antigens would beproduced. As still another example, the immunogen may be an allergenthat increases a T_(H)1 type response and hence increase production oftypical T_(H)1 cytokines such as y-interferon (IFN-γ), tumor necrosisfactor-β(TNF-β), and interleukin-2 (IL-2) which, in turn, may decreaseinflammation in allergic conditions such as asthma.

[0046] The invention will be further appreciated in light of thefollowing examples.

EXAMPLE 1

[0047] Tumor Cell Lines

[0048] The EL4 thymoma cell line (TIB-39) was obtained from AmericanType Culture Collection (ATCC, Rockville, Md.). The cells weremaintained in culture using RPMI 1640 medium supplemented with 10% fetalcalf serum (FCS) (HyClone Laboratories, Logan, Utah), 15 mM HEPESbuffer, 2 mM glutamine, 0.1 mM non-essential amino acids, 50 units/mlpenicillin, 50 units/ml streptomycin, 1 mM sodium pyruvate (Biofluids,Rockville, Md.), and 50 μM 2-mercaptoethanol (Sigma, St. Louis, Mo.).

[0049] Antigens

[0050] Purified chicken egg ovalbumin (OVA) (grade V) was purchased fromSigma (St. Louis, Mo.). The H-2Kb restricted peptide epitope of OVAprotein, OVA₂₅₇₋₂₆₄ (SIINFEKL), was synthesized using FMOC chemistry onan Applied Biosystems Model 432A peptide synthesizer. The lyophilizedproduct was resuspended in water at a concentration of 2 mg/ml, sterilefiltered and stored at −70° C. The peptide was determined by highperformance liquid chromatography to be greater than 90% pure.

[0051] OVA protein was coated onto inert particles and the antigen wasencapsulated using an aqueous enteric coating system containing abiodegradable polymethacrylic acid copolymer (Eudragit L30D). The inertparticles were NuPareils® measuring about 45 mesh.

[0052] Immunization

[0053] Six- to eight-week-old C57BL/6 (H-2K^(b)) female mice wereobtained from Taconic Farms (Germantown, N.Y.). These animals wereimmunized either by subcutaneous injection with 30 μg OVA proteinemulsified in DETOX-PC® adjuvant (RIBI ImmunoChem Research, Hamilton,Mont.), or orally via intubation into the back of the throat withmicrospheres containing 200 μg OVA. Control mice were orally fed aplacebo microsphere. A series of three immunizations was performed ondays 0, 14, and 28. Animals were euthanized three weeks following thefinal immunization.

[0054] Lymphoproliferation

[0055] Spleens were removed from immunized animals 21 days after theirthird immunization and were mechanically dispersed through a 70 μm nyloncell strainer (Falcon; Becton Dickinson, Franklin Lakes, N.J.) to yielda single cell suspension. Dead cells and erythrocytes were removed bycentrifugation over a Ficoll-Hypaque gradient (d=1.119 g/cm). Therecovered cell population was then enriched for T cells by passing thesplenic mononuclear cells over nylon wool columns (Robbins ScientificCorp., Sunnyvale, Calif.). The enriched T cells were washed in completemedium (RPMI 1640 supplemented with 10% FCS, 15 mM HEPES buffer, 2 mMglutamine, 0.1 mM non-essential amino acids, 50 units/ml penicillin, 50units/ml streptomycin, 50 μM 2-mercaptoethanol, and 1 mM sodiumpyruvate) and dispersed into 96-well flat-bottom microtiter plates(Falcon; Becton Dickinson, Lincoln Park, N.J.) at a concentration of1×10⁵/well.

[0056] The T lymphocytes were then incubated in the presence of naivesyngeneic splenocytes (5×10⁵/well) as antigen presenting cells (APC).Stimulated wells contained either OVA protein (100,μg/ml), OVA₂₅₇₋₂₆₄peptide (100 μg/ml), or concanavalin A (Con A; 2.5 μg/ml). Control wellscontained only T cells and APC in complete medium. All cultures were ina final volume of 200 pi and were incubated at 37° C. in 5% CO₂ foreither 2 days (Con A) or 5 days (antigen stimulants). Cultures werepulsed with 1 μCi/well [³H]thymidine (DuPont New England Nuclear,Wilmington, Del.) for the final 18 to 24 hours. Cultures were harvestedusing a PHD cell harvester (Cambridge Technology, Cambridge, Mass.) andincorporated radioactivity was quantitated by liquid scintillationspectroscopy (LS 60001C, Duarte, Calif.). The results of triplicatewells were averaged and are reported as a stimulation index (SI)calculated by the following formula:

SI=stimulated wells (cpm)/control wells (cpm)

[0057] In vitro Stimulation of CTL

[0058] Primary CTL Cultures

[0059] Splenocytes (25×10⁶) harvested from each experimental group,pooled from the spleens of three animals per group, were incubated in 10ml of complete RPMI (10% FCS, 15 mM HEPES buffer, 2 mM glutamine, 0.1 mMnon-essential amino acids, 50 units/ml penicillin, 50 units/mlstreptomycin, 50 μM 2-mercaptoethanol, and 1 mM sodium pyruvate) inupright 25 cm² flasks at 37° C. in 5% CO₂ in the presence of 5 μg/mlOVA₂₅₇₋₂₆₄ peptide. Long-term CTL Lines

[0060] Primary CTL cultures were harvested after seven days, and viablelymphocytes were recovered by centrifugation over a Ficoll gradient(d=1.08 g/ml; Organon Teknika Corp., Durham, N.C.). The recovered cellswere restimulated in 24-well flat-bottom plates (Corning Costar Corp.,Cambridge, Mass.) containing 0.5×10⁶ lymphocytes, 5×10⁶ irradiated(2,000 rads) syngeneic C57BL/6 spleen cells, 5 μg/ml OVA₂₅₇₋₂₆₄ peptide,and 10 units/ml recombinant human interleukin-2 (IL-2) (Cetus Corp.,Emeryville, Calif.). Subsequent weekly restimulations of antigenspecific CTL were performed in the same manner with the exception ofpeptide dose. After 8 weeks of in vitro stimulation, the peptideconcentration was reduced to 2 μg/ml.

[0061] Cytotoxicity Assays

[0062] Four hour ⁵¹Cr release assays were performed. Target cells (tumorcells) were labeled with 50 μCi Na⁵¹CrO₄/1×10⁶ cells for 90 minutes.Target cells (1×10⁴) were labeled in 50 μl of complete RPMI medium andwere added to the wells of a 96-well U-bottom plate (Corning CostarCorp.). When appropriate, target cells were incubated for 30 minutes at37° C. in 5% CO₂ with one or more of the following before the additionof T cell effectors: OVA₂₅₇₋₂₆₄ peptide, anti-CD8 antibody (supernatantfrom the 2.43 hybridoma), or anti-CD4 antibody (supernatant from the GK1.5 hybridoma). Effector cells were added to the targets in 50 μl ofcomplete medium. The plates were then incubated at 37° C. in 5% CO₂ forfour hours. Following incubation, supernatants were harvested usingSkatron harvesting frames (Skatron, Inc., Sterling, Va.). The release ofradioactivity was quantitated using a gamma counter (BeckmanInstruments) and the percent specific lysis was calculated using theequation:${\% \quad {specific}\quad {lysis}} = {\frac{{{experimental}\quad ({cpm})} - {{spontaneous}\quad {release}\quad ({cpm})}}{{{maximum}\quad {release}\quad ({cpm})} - {{spontaneous}\quad {release}\quad ({cpm})}} \times 100}$

[0063] Results were reported as the mean plus or minus the standarderror of the mean of triplicate cultures.

[0064] Spontaneous release was calculated from wells to which 100 ml ofmedium had been added in the absence of T cell effectors. Maximumrelease was calculated from wells to which a solution of 2% Triton X-100was added. Flow Cytometry

[0065] Lymphocytes were harvested and washed three times with coldDulbecco's phosphate-buffered saline (DPBS) containing Ca²⁺ and Mg²⁺supplemented with 5% fetal bovine serum (FBS). Cells were incubated onice for 45 minutes with either fluorescein isothiocyanate(FITC)-conjugated anti-mouse CD2, CD3, CD4, CD8, CD28, CD11a/CD18, andα/βT cell receptor (TCR), or the appropriate isotype controlFITC-conjugated rat IgG2aK, rat IgG2bλ, or hamster IgG antibody(PharMingen, San Diego, Calif.), and then washed twice with DPBSsolution free of Ca²⁺ and Mg²⁺. Data from 10,000 live cells/sample wereanalyzed using flow cytometric analysis as known to one skilled in theart with a Becton Dickinson FACScan® flow cytometer using an excitationwavelength of 488 nm and a band pass filter of 530 nm.

[0066] Lymphoproliferative Analysis

[0067]FIG. 2A and FIG. 2B show the results of a lymphoproliferativeanalysis. As shown in FIG. 2A, and to determine if oral immunizationwith the model protein OVA could result in the activation of a cellularimmune response, C57BL/6 mice were immunized three times withenterocoated microspheres containing OVA protein (microsphere-OVA) atconcentrations of 12.5 μg/ml, 25 μg/ml, 50 μg/ml and 100 μg/ml (hatchedbars). To compare the immune response generated following oralimmunization with OVA to that of parenteral immunization with the sameantigen, OVA protein was emulsified in DETOX-PC® adjuvant andadministered subcutaneously to a second group of C57BL/6 mice (solidbars). A third group of C57BL/6 mice received a placebo microsphere byoral administration (open bars). Lymphocyte proliferation was assessedby measuring [³H]thymidine incorporation.

[0068] As shown in FIG. 2A, T cells from mice receiving 100 μg/mlmicrosphere-OVA orally had a stimulation index of 38.3, while T cellsfrom mice immunized with OVA protein in adjuvant had a stimulation indexof 9.1. Naive splenocytes did not proliferate in the presence of OVAprotein. As shown in FIG. 2B, lymphocytes from each group showed strongstimulation indices upon non-specific mitogen stimulation with 2.5 μg/mlCon A.

EXAMPLE 2

[0069] A CTL immune response in mice that had been orally immunized withenterocoated microsphere-OVA generated an antigen-specific T cell line.Purified splenocytes from mice immunized with OVA, either orally inmicrospheres or, as a control, subcutaneously in an emulsion withDETOX-PC® adjuvant, were cultured in vitro in the presence of OVA₂₅₇₋₂₆₄peptide, irradiated syngeneic splenocytes as APC, and IL-2. The celllines were maintained on seven-day cycles of in vitro stimulation (IVS).The ability of the cell lines to lyse target cells in anantigen-dependent manner was evaluated five days into the IVS cycleusing a four-hour ⁵¹Cr release assay. The EL4 (H-2K^(b)) cell line wasused as a target cell in these assays. EL4 cells were pre-pulsed withOVA₂₅₇₋₂₆₄ peptide prior to the addition of T cell effector cells intothe assay. All data are at a 20:1 effector:target ratio.

[0070] As shown in FIG. 3A and FIG. 3B, the emergence ofantigen-specific lysis was evident after only three cycles of IVS. FIG.3A shows T cell effectors from mice immunized by subcutaneousadministration of OVA emulsified in DETOX-PC® adjuvant. FIG. 3B shows Tcell effectors from mice immunized by oral administration ofmicrosphere-OVA. Closed circles represent EL4 cells pre-pulsed with 25μg/ml OVA₂₅₇₋₂₆₄ CTL epitope peptide. Open circles represent non-pulsedEL4 cells.

[0071] While antigen-specific lysis was evident after three cycles ofIVS, non-specific lysis of EL4 cells was also observed at this timepoint. Following six cycles of IVS, non-specific lysis of EL4 cells haddropped substantially (about 10% to about 20%). At the eighth cycle ofIVS, both cell lines were approaching higher (about 50% to about 60%)levels of antigen-specific lysis with very low levels (less than about10%) of non-specific lysis.

[0072] As shown in FIG. 4, the strength of the CTL lines derived fromimmunized animals was evaluated as a function of effector:target ratio.EL4 cells were pre-pulsed with 25 μg/ml OVA₂₅₇₋₂₆₄ peptide. Closedcircles represent microsphere-OVA. Closed squares represent OVAemulsified in DETOX-PC® adjuvant. Crosses represent placebo microspheresand open triangles represent non-specific ⁵¹Cr uptake of non-peptidepulsed EL4 cells.

[0073] Both CTL lines could be titrated through a range ofeffector:target ratios. When splenocytes from animals that had beenadministered placebo microspheres were cultured under the sameconditions as the experimental cell lines, they could not be in vitroactivated to recognize peptide pulsed target cells. This observationalso demonstrated that the experimental cell lines acquired theirantigen specificity via in vivo activation following oral or parenteralimmunization with OVA, and not as a result of in vitro cultureconditions.

[0074] To confirm that the cell lines derived from each group ofimmunized animals lysed tumor cells in a CD8+ T cell dependent fashion,antibody blocking experiments were performed. FIG. 5A and FIG. 5B showCD8+ T cell dependence of antigen-specific target cell lysis. FIG. 5Ashows a four hour ⁵¹Cr release assay at a 40:1 effector:target ratiousing OVA₂₅₇₋₂₆₄ pulsed EL4 target cells, to determine dependence ofCD8+ T cells on the observed target cell lysis by the CTL line derivedfrom animals immunized by subcutaneous administration of OVA inadjuvant. FIG. 5B shows a four hour ⁵¹Cr release assay at a 20:1effector: target ratio using OVA₂₅₇₂₆₄ pulsed EL4 target cells, todetermine dependence of CD8+ T cells on the observed target cell lysisby the CTL line derived from animals immunized by oral administration ofmicrosphere-OVA.

[0075] As shown in FIG. 5A and FIG. 5B, in four hour ⁵¹Cr releaseassays, the supernatant from either the hybridoma GK1.5, secretinganti-CD4 antibody, or the hybridoma 2.43, secreting anti-CD8 antibody,was incubated with T cells prior to their addition to OVA₂₅₇₋₂₆₄ pulsedEL4 target cells. In the presence of anti-CD8 antibody, theantigen-specific tumor cell lysis was inhibited. Conversely, thepresence of anti-CD4 antibody resulted in minimal (about 1% to about10%) inhibition of T cell mediated antigen-specific cell lysis. Thelytic activity of both T cell lines was eliminated when the T cells werepre-incubated with the supernatant of the 2.43 hybridoma that containsanti-CD8 antibody. Preincubation of the T cells with GK 1.5 hybridomasupernatant containing anti-CD4 antibody did not cause a major decreasein the lytic activity of the cell line.

[0076] FACS Analysis

[0077] The presence of T cell surface markers on OVA-derived cell lineswas analyzed by flow cytometry. Table 1 shows phenotypiccharacterization of T cell lines following eight cycles of IVS. The celllines were derived from splenocytes of mice that had been immunized witheither microsphere-OVA or OVA in adjuvant as previously described. TABLE1 % Positive Cells Cellular (mean fluorescence intensity) DeterminantOvalbumin-DETOX-PC ® Microsphere Ovalbumin CD3 95.55 (23.32) 99.18(77.94) CD4 57.69 (62.37)  8.02 (52.63) CD8  49.68 (138.16)  92.69(174.30) CD2 78.82 (19.98) 69.14 (26.37)  CD28 12.98 (32.01) 60.99(18.38) CD11a/CD18  99.58 (157.77) 98.22 (89.00) α/β TCR 64.34 (16.53)77.33 (21.78)

[0078] As shown in Table 1, both cell lines had a population of greaterthan about 95% T cells as identified by the CD3 cell surface molecule.The T cell line derived from lymphocytes cultured from mice immunizedwith OVA in adjuvant contained 49.6% CD8⁺ T cells, and the cell linederived from lymphocytes cultured from mice orally immunized withmicrosphere-OVA contained 92.7% CD8⁺ T cells. Both cell lines were shownto express the costimulatory molecule receptors CD2 and CD28, inaddition to the integrin molecule CD11a/CD18. The cultured T cells fromboth groups of immunized animals also expressed the usage of an α/β Tcell receptor. These data help to illustrate that the T cells activatedthrough oral microsphere immunization with the protein antigen OVA arephenotypically similar to the repertoire activated following parenteralimmunization with the same antigen.

[0079] The microspheres of the present invention modulate an immuneresponse. The response may encompass a general enhanced production ofT_(H)1 cells, T_(H)2 cells and cytotoxic T lymphocyte (CTL) subsets, oran enhanced shift from a T_(H)2 type response to a T_(H)1 type response,or an enhanced shift from a T_(H)1 type response to a T_(H)2 typeresponse, or an enhanced differentiation of pre-CTL to CTL. Theimmunogen may be a peptide, a protein fragment, a protein, a DNA, and/oran RNA, and may be a gene, a gene fragment or a vaccine. The therapeuticor prophylactic agents encompass immunogens, immunotherapy agents orgene therapy agents, either separately or in combination, that may beorally delivered in enteric microencapsulated formulations as bound toan inert particle having a size greater than about 35 mesh and in theform of a substrate bead, granule, powder, or crystal.

[0080] It will be appreciated that the delivery system composition andmethods disclosed herein can be used prophylactically andtherapeutically in a wide array of conditions. Thus, the embodiments ofthe present invention shown and described in the specification are onlypreferred embodiments of the inventor who is skilled in the art and arenot limiting in any way. Various changes, modifications or alterationsto these embodiments may be made or resorted to without departing fromthe spirit of the invention and the scope of the following claims.

What is claimed is:
 1. A method of inducing an immune response in amammal comprising administering a microsphere comprising an immunogenbound to an inert particle to a small intestine of said mammal, saidinert particle having a mesh size greater than about 35 mesh.
 2. Themethod of claim 1 wherein said microsphere is administered orally andsaid microsphere comprises an enteric coated microsphere.
 3. The methodof claim 1 wherein said microsphere is administered in a gel capsule. 4.The method of claim 1 wherein said immunogen is selected from the groupconsisting of a peptide, a protein fragment, a protein, a gene, a genefragment, a DNA, an RNA and combinations thereof.
 5. The method of claim1 wherein said immunogen is a vaccine.
 6. The method of claim 1 furthercomprising administering a potentiating agent bound to an inertparticle, said inert particle selected from the group consisting of animmunogen-bound inert particle and a non-immunogen bound inert particle.7. The method of claim 1 wherein a plurality of microspheres areadministered to selectively induce the immune response.
 8. The method ofclaim 7 wherein said microspheres have compositions selected from thegroup consisting of different inert particle sizes, different inertparticle compositions, different enteric coatings, differentformulations and combinations thereof.
 9. The method of claim 1 whereinsaid microsphere containing said immunogen induces an increase in thenumber of T lymphocytes.
 10. The method of claim 9 wherein saidmicrosphere containing said immunogen induces an increase in a cellpopulation selected from the group consisting of a T_(H)1 lymphocyte, acytotoxic T lymphocyte (CTL), and combinations thereof.
 11. The methodof claim 1 wherein said inert particle has a mesh size greater thanabout 40 mesh.
 12. The method of claim 1 where said immunogen iscontained on an inert particle selected from the group consisting of anonpareil, a silica powder, a salt crystal and a sugar crystal.
 13. Amethod of treating cancer in a mammal comprising administering amicrosphere comprising an immunogen bound to an inert particle to asmall intestine of said mammal, said inert particle having a mesh sizegreater than about 35 mesh.
 14. The method of claim 13 wherein saidmicrosphere is administered orally and said microsphere comprises anenteric coated microsphere.
 15. The method of claim 13 wherein saidmicrosphere is administered in a gel capsule.
 16. The method of claim 13wherein said immunogen is selected from the group consisting of apeptide, a protein fragment, a protein, a gene, a gene fragment, a DNA,an RNA and combinations thereof.
 17. The method of claim 13 wherein saidimmunogen is a vaccine.
 18. The method of claim 13 further comprisingadministering a potentiating agent bound to an inert particle, saidinert particle selected from the group consisting of an immunogen-boundinert particle and a non-immunogen bound inert particle.
 19. The methodof claim 13 wherein a plurality of microspheres are administered toselectively induce the immune response.
 20. The method of claim 19wherein said microspheres have compositions selected from the groupconsisting of different inert particle sizes, different inert particlecompositions, different enteric coatings, different formulations andcombinations thereof.
 21. The method of claim 13 wherein saidmicrosphere containing said immunogen induces an increase in the numberof T lymphocytes.
 22. The method of claim 21 wherein said microspherecontaining said immunogen induces an increase in a cell populationselected from the group consisting of a T_(H)1 lymphocyte, a cytotoxic Tlymphocyte (CTL), and combinations thereof.
 23. The method of claim 13wherein said inert particle has a mesh size greater than about 40 mesh.24. The method of claim 13 wherein said immunogen is contained on aninert particle selected from the group consisting of a nonpareil, asilica powder, a salt crystal and a sugar crystal.
 25. A method ofinducing an immune response in a mammal comprising orally administeringto said mammal a microsphere comprising an enteric-coated inert particlecontaining a protein immunogen, said particle having a mesh size greaterthan about 40 mesh.
 26. The method of claim 25 further comprisingadministering a potentiating agent bound to an inert particle, saidinert particle selected from the group consisting of an immunogen-boundinert particle and a non-immunogen bound inert particle.
 27. The methodof claim 25 wherein a plurality of microspheres are administered toselectively induce the immune response.
 28. The method of claim 27wherein said microspheres have compositions selected from the groupconsisting of different inert particle sizes, different inert particlecompositions, different enteric coatings, different formulations andcombinations thereof.
 29. The method of claim 25 wherein the immuneresponse comprises an increase in a T lymphocyte population.
 30. Themethod of claim 29 wherein the immune response comprises an increase ina cell population selected from the group consisting of a T_(H)1lymphocyte, a cytotoxic T lymphocyte (CTL), and combinations thereof.31. A composition adapted to induce an immune response comprising animmunogen contained on an inert particle and having an enteric coating,said inert particle having a mesh size greater than about 35 mesh. 32.The composition of claim 31 contained in a gel capsule.
 33. Thecomposition of claim 31 further comprising a potentiating agent.